(a) Field of the Invention
The invention relates to methods for enhancement of naturally occurring cytoplasmic male sterility and for restoration of male fertility and uses thereof in hybrid crop production.
(b) Description of Prior Art
For many crops, hybrids, formed by crossing two or more different strains, provide higher yields than do the parental strains themselves. For example, in canola (Brassica napus, campestris), Canada's most important crop, manually produced hybrids have yields that can be up to 50% greater than those of the parental lines. This phenomenon, termed hybrid vigor, has been most successfully applied in maize (Zea mays). Hybrid maize constitutes approximately 90% of the North American crop.
To produce hybrid seed on a commercial scale, it is necessary to prevent self-pollination of the seed parent of the hybrid cross. This is a relatively simple matter in maize, because the male or pollen-producing organs (the stamens) are located in a different part of the plant than the female, or seed-bearing organs (the carpels), and the stamens, which collectively form a structure called the tassel, can easily be removed manually from the large numbers of plants used in a seed production operation. By contrast, in most crop species, such as canola, the stamens and carpels are located in the same floral structure, and manual emasculation of large numbers of plants is not possible. Hence, seed producers require alternative methods of pollination control for producing hybrids in these crops. A review of the technology developed for this purpose can be found in The PBI Bulletin article of Arnison P. (Arnison P. et al., The PBI Bulletin, January 1997, 1-11).
One alternative method is to use chemicals, called gametocides, that specifically kill pollen. These chemicals are generally expensive and are often only partially effective. For most crops, especially those like canola that flower over an extended period, this type of pollination control is not cost-effective. Nearly all hybrid seed production systems, therefore, rely on genetic control of pollination. Genetic pollination control mechanisms fall into three categories: self-incompatibility, “molecular hybridization” methods, and cytoplasmic male sterility.
Self incompatibility (SI) results from the capacity of some plants to reject their own pollen. Plants expressing SI can be used as the female line in hybrid production, but, because these plants normally reject their own pollen, it is usually difficult, and cost-ineffective, to propagate or maintain such lines. In addition, self-incompatibility is not found in most plant species. Molecular hybridization methods are the most recently developed. They rely on genetically engineered male sterility, caused by the specific expression of toxic proteins in pollen forming cells. Such introduced toxin genes act as dominant male sterility genes and can be used to generate female lines for hybrid seed production. As with SI, the propagation of such female lines is not straightforward and involves loss of plant material. The usefulness of these methods is still unclear, and at present they are used in the production of only a few hybrid varieties which are not widely grown.
Cytoplasmic male sterility (CMS), is a widespread and classic non-Mendelian trait. CMS plants are incapable of self-pollination and hence when a CMS line is planted alongside a male-fertile line, all the seed that forms on the sterile plants will be a hybrid of the two parents. Unlike most traits, CMS is maternally transmitted, i.e., it is passed on to offspring only through the seed parent. This property results from the fact that the gene or genes that determine CMS are located on mitochondrial DNA (mtDNA). Unlike most genes, which reside in nuclear DNA, genes in mtDNA are transmitted solely through the female in most plant species. As a result of this property of maternal transmission, it is possible to easily propagate female CMS lines, by pollinating with a male fertile “maintainer” line, that is identical to the CMS line in its nuclear genes, but which is male fertile because it lacks the CMS-causing mtDNA. However, again as a result of the maternal transmission of CMS, hybrid plants produced using female CMS lines would also be male sterile because they would carry the male-sterility conferring mtDNA. This is problematic for seed crops such as maize and canola, which require pollen production for the formation of seed, the harvested product. Fortunately, in many crop species, specific dominant nuclear genes termed restorers of fertility (Rf) have been identified that can suppress the male-sterile phenotype and “restore” fertility to F1 hybrids. The components of a CMS system, therefore, consist of the CMS line (which contains the male sterile (S) cytoplasm (or mtDNA) and is homozygous for the recessive or maintainer allele of the restorer gene), the maintainer line (which contains a fertile or normal mtDNA (F) but is isogenic with the CMS line at nuclear genetic loci), and the restorer line (which usually contains the male sterile mtDNA but is homozygous for the dominant nuclear Rf gene). The use of these components in a hybrid seed production scheme is illustrated in FIG. 1.
To produce a diverse set of hybrids using CMS, adequate numbers of restorer lines, that contain Rf genes, as well as “maintainer” lines, that are sterilized by the CMS mtDNA, must be available. The development of these lines through conventional genetics is a slow process that minimally requires several years of effort. For example, to develop a new restorer line it is necessary to first cross the recipient line with an existing restorer line to introduce the Rf allele. A series of backcrosses are then required to recover the genotype of the recipient line. Even after many generations of backcrosses some donor DNA linked to the Rf gene will remain, a phenomenon termed linkage drag; this donor DNA may carry deleterious traits and compromise the quality of the recipient strain.
Two CMS systems are proving to have some limited use in hybrid canola seed production. The Polima or pol cytoplasm is capable of conferring male sterility on many canola cultivars, and effective restorer lines, possessing dominant alleles at a single nuclear restorer locus, have been identified. The cost-effectiveness of hybrid production using this system is limited by the fact that pol-induced male sterility tends to be incomplete or “leaky”, especially when the female line is exposed to warmer growing conditions. As a result, pol hybrid seed may be contaminated with CMS seeds that result from self-pollination of the female line. As these CMS plants are male sterile and do not spontaneously set seed, their presence can considerably lower the overall yield of the “hybrid” mixture. A second CMS system is based on the use of a hybrid cytoplasm in which the male sterility determinant is derived from a radish cytoplasm termed Ogura or ogu. Male sterility conferred by the ogu cytoplasm is complete. Restorer lines for this system, which were developed by introgressing a single radish restorer gene (which we designate here as Rfo) into Brassica napus, have recently become available, but the development of restorer lines is hampered by “linkage drag” between the restorer gene and genes that have a negative effect on seed quality.
It would be highly desirable to be provided with methods for enhancement of naturally occurring cytoplasmic male sterility and for restoration of male fertility and uses thereof in hybrid crop production.